Soybean [Glycine max (L.) Merr.] is one of the world""s most important agronomic crops. Between 120 and 130 million acres are planted annually, resulting in 105 million tons of seed. Soybeans have dominated world oilseed production among the eight major oilseeds traded in international markets, accounting for over 97% of all world oilseed production since 1965. The value of the crop is estimated to be over 20 billion dollars. Both soybean oil and protein are used extensively in food products for human consumption. In the United States, 5% of the total protein is derived from grain legumes and up to 65% of the oil used by the food processing industry comes from soybean (Hoskin, 1987; Smith and Huyser, 1987).
Although a great deal of effort has been devoted towards the development of new cultivars of soybean with improved disease resistance, along with increased nutritional value, traditional breeding programs have been restricted because soybean germplasm is extremely narrow and the majority of the soybean cultivars in use are derived from very few parental lines (Christou et al., 1990).
Hence, modification of soybean using genetic engineering techniques would facilitate the development of new varieties with traits such as disease resistance, e.g., viral resistance, pest resistance, and herbicide resistance, and seed quality improvement in a manner unattainable by traditional breeding methods or tissue-culture induced variation. To attain genetically modified plants, a transformation system must be developed to optimize the integration of DNA in the plant, which is most commonly delivered using either an Agrobacterium-based system, which requires wounding of plant cells (Zambryski et al., 1989), or particle bombardment (Biolistics). Although transgenic soybean plants have been produced using both microprojectile bombardment (McCabe et al., 1988; Christou, P. et al., 1989) and various Agrobacterium-mediated transformation methods (Hinchee et al., 1988; Chee et al., 1989; Parrott et al., 1989; Clemente and Zhang, 2000; Di et al., 1996), legumes, including soybeans remain extremely recalcitrant to transformation (Trick, 1997). And while successes in producing transgenic plants have been reported, the frequency of attaining transgenic plants is low, e.g., Parrott et al. (1994) report 1 transgenic plant out of 195 regenerated, and Zhang et al. (1999) report that the efficiency of producing marker-positive plants in five independent attempts was 0%, 0%, 0.5%, 0.7% and 3.0%. The demand and need for new and useful transgenic soybeans is evident from the fact that transgenic soybeans, which were derived from a single transgene integration event, represent more than 50% of the total commercial production of soybeans grown in the United States. In addition, the recalcitrant nature of soybeans to transformation has rendered many molecular, genetic, and genomic techniques commonly used in other major crops, such as maize, impractical.
The xe2x80x9ccot-nodexe2x80x9d method is a frequently used soybean transformation system based on Agrobacterium-mediated T-DNA delivery into regenerable cells in the cotyledonary node. For example, U.S. Pat. No. 5,322,783 relates to a method for transformation of soybean tissue in which cotyledonary node cells are treated with a cytokinin, and then the cells are bombarded with microparticles carrying specific vectors and exogenous DNA. U.S. Pat. Nos. 5,169,770 and 5,376,543 disclose a method in which soybean seeds are germinated, and the meristematic or mesocotyl cell tissues are inoculated with bacterial cells, specifically Agrobacterium strains which, through infection, transfer DNA into these explants.
In U.S. Pat. No. 4,992,375, a process is described in which the cotyledonary node region from a donor plant is excised, and the explant is cultured in a nutrient media containing cytokinin, until shoots arose from resultant callus. The shoots are then rooted. U.S. Pat. No. 5,416,011 also utilizes a cotyledon explant, which requires removal of the hypocotyl, saving and separating the cotyledons, and inserting a chimeric gene by inoculation with Agrobacterium tumefaciens vectors containing the desired gene. The histochemical marker GUS was employed to determine successful transformation. Nevertheless, the efficiency of the cot-node transformation system remains low apparently because of poor Agrobacterium infection of cot-node cells, inefficient selection of transgenic cells that give rise to shoot meristems, and low rates of transgenic shoot regeneration and plant establishment.
A number of reports on soybean regeneration utilized cotyledons from immature zygotic embryos induced to undergo somatic embryogenesis (Liu et al., 1992). Soybean regeneration through short meristem cultures resulted in up to 35% explants responding to plant regeneration 4 weeks after culture (Kartha et al., 1981). Regeneration via organogenesis utilizing different explants has been reported with a maximum of 97% of explants responding and 3 shoots produced per explant 10 weeks after culture, and 38% of shoots developing roots for another 4 weeks (Yeh et al., 1991). However, interactions between genotype and in vitro cultural conditions (medium, explant and light treatment) have not been reported in regeneration via organogenesis or meristem culture in soybean, although it has been studied in regeneration via somatic embryogenesis and was proven important (Powell et al., 1987; Komatsuda et al., 1991).
The unreliable transformation and regeneration of legumes in general is due, in part, to the difficulty in producing fertile mature plants from tissue culture as well as legumes being extremely resistant to Agrobacterium infection. Thus, although genes have been transferred to soybean protoplasts by electroporation of free DNA (Christou et al., 1987; Lin et al., 1987), regeneration technology for soybean has not progressed to a state where regenerated plants can be produced from protoplasts. For example, the formation of shoots, and eventual rooting, takes place only in a tiny fraction of the cases. Further, successful transformation and successful regeneration are frequently cultivar-specific, with no broad success. See, for example, Wayne et al., 1988; Finer et al., 1991; Sato et al., 1993; Moore et al., 1994; Parrott et al., 1994 and Steart et al., 1996.
Improvements have been reported in the three components of the cot-node transformation system. For example, improved selection systems and plant regeneration have been developed (Zhang et al., 1999). Considerable effort also has been applied to increasing Agrobacterium virulence by addition of chemical inducers of the vir genes (Bolton et al., 1986; Dyxc3xa9 et al., 1997), improvements in vir gene constructs (Hansen et al., 1994; Torisky, 1997), identification and selection of susceptible soybean cultivars (Meuer et al., 1998; Byrne et al., 1987; Delzer et al., 1990; Cho et al., 2000), and increasing the wounding of explants by either microprojectile bombardment or sonication (Bidney et al., 1992; Santarem et al., 1998).
Although agents such as dithiothreitol (DTT) and polyvinylpolypyrrolidone (PVPP) increase plant viability after Agrobacterium-mediated transformation of grape (Perl et al., 1996) and ascorbic acid, the amino acid, cysteine, and silver nitrate individually or in combination decreased damage and increased viability of Japonica rice meristem cultures and, in combination, decreased the Agrobacterium-mediated tissue necrosis of those cultures (Enrxc3xadguez-Obregxc3x3n et al., 1999), no agents have been reported to enhance the Agrobacterium-mediated transformation efficiency of soybeans.
Thus, what is needed is a method to reproducibly enhance the transformation of plants, e.g., soybeans.
The invention provides a method for transforming a plant cell, part or tissue. Preferred plant cells, parts or tissue for use in the method of the invention are those which can be regenerated to a plant. The method comprises contacting a plant cell, part or tissue, e.g., a cotyledon explant from a plant seedling, with a Agrobacterium, e.g., A. tumefaciens or A. rhizogenes, containing DNA to be introduced into the plant cell, part or tissue and at least one agent in an amount that enhances Agrobacterium-mediated transformation so as to yield a transformed plant cell, part or tissue. Then a transformed plant cell, part or tissue is identified. Preferably, the plant cell, part or tissue is wounded prior to contact. For example, for a cotyledonary explant, the cotyledon is wounded in the region of the axillary bud and/or cotyledonary node. The cotyledon may be prepared by (i) removing the hypocotyl region of a seedling by cutting in the region just below the cotyledonary node, for example, from about 0.2 to about 1.5 cm below the cotyledonary node; (ii) splitting and completely separating the remaining attached hypocotyl segment, also thereby separating the two cotyledons; and (iii) removing the epicotyl from the cotyledon, e.g., to which it remains attached. Prior to removing the hypocotyl region, the seedling may be incubated at about 0xc2x0 C. to about 30xc2x0 C., e.g., 0xc2x0 C. to about 10xc2x0 C. or 15xc2x0 C. to about 30xc2x0 C., for at least 24 hours. Preferably, the seedling is a 5-day germinated seedling that is bisected between the cotyledons along the embryonic axis. The epicotyl is excised and the cot-node cells are wounded with a scalpel by extensive cutting of the node at the base of the cotyledon. Then the wounded cotyledon is contacted with a Agrobacterium vector, e.g., a disarmed A. tumefaciens vector containing DNA, the cot-node explants are cultivated on solid medium for 5 days and transformed explant tissue is identified, e.g., by selection. Sources of the plant cell, plant part, or plant tissue include both dicots and monocots, including agricultural crops, ornamental fruits, vegetables, trees and flowers. In one embodiment, the plant cell, part or tissue is that of a legume. Preferably, a differentiated transformed plant is regenerated from the transformed plant cell, part or tissue.
Preferred agents for use in the methods of the invention include, but are not limited to, those which inhibit enzymatic browning of plant tissue, plant cells, or parts of a plant, in response to wounding, e.g., an agent that inhibits the activity or production of enzymes associated with browning such as polyphenol oxidase (PPO) and peroxidase (POD), chelators of metals required for activity of the enzymes associated with browning, as well as sulfhydryl-containing agents, e.g., cysteine, L-cystine, DTT, ascorbic acid, sodium thiosulfate, and glutathione.
As described hereinbelow, the Agrobacterium-mediated infection of soybean explants in the cot-node region was increased from 30% to 100% by employing an agent of the invention and the following general protocol. Under aseptic conditions, the axillary region near the node located between the cotyledon and hypocotyl of 5-day old soybean seedlings was excised. The explant tissued was dissected from the entire seedling by cutting the hypocotyl approximately 0.5 cm to 1 cm below the cotyledon and cutting lengthwise down the hypocotyl resulting in two separate explants. After the epicotyl was removed, the entire node region, including the axillary region, was wounded with a scalpel, and the explant was co-cultivated in a liquid Agrobacterium culture before placing the explant on a solid co-cultivation media for 5 days. For example, Agrobacterium strain AGL1 and a binary plasmid BSF16 that contains the bar gene for herbicide (PPT) selection, the xcex2-glucoronidase (GUS) gene for a phenotypic marker, and a sulfur-rich gene, albumin, from sunflower driven by a seed-specific promoter, was employed. De novo shoot formation occurred at the site of the axillary meristem when grown in a shoot induction media under herbicide selection after four weeks. After this time, elongation of herbicide-resistant shoots was induced for up to ten weeks on a shoot elongation media.
Surprisingly, the addition of the sulfhydryl compound L-cysteine to the co-cultivation media during the 5-day incubation step increased the amount of GUS+ sectors at the cot-node region dramatically. For example, Agrobacterium was suspended in the liquid co-culture for about 1 hour to about 2 hours and then the wounded explant was added to the Agrobacterium liquid co-culture for about one half of an hour. The explants were then placed on solid co-culture media for 5 days. The Minnesota genotypes Bert, MN1301, and MN0901 were employed with either 0 mg/l, 100 mg/l, 200 mg/l, 300 mg/l, or 400 mg/l L-cysteine. Transient assay experiments after the 5-day incubation period resulted in 80-100% infection (% of explants) at the entire cot-node region, the appearance of GUS+ foci on the cotyledon, as well as extensive GUS+ foci along the cut hypocotyl surface, in explants contacted with cysteine containing media. Similar results were observed with the strain LBA4404 containing the pTOK233 binary plasmid. Generally, in the absence of cysteine, only 50% of control explants showed infection in the cot-node region and at a much reduced frequency. It is also very rare to detect GUS+ foci on the cotyledon tissue.
As further described hereinbelow, under a low selection pressure (1.6 to 3.33 mg/l PPT), the control (0 mg/l cysteine) on average had 3.3 GUS+ foci/explant scored, while explants co-cultivated in 400 mg/l cysteine had an average of 15.6 GUS+ foci/explant scored after 4 weeks of shoot initiation. Moreover, increasing selection pressure during shoot induction may also increase the number of GUS+ shoots. Plants co-cultivated in 0 mg/l cysteine or 400 mg/l cysteine were placed in shoot induction media containing either 5 mg/l or 3.33 mg/l PPT. The results were as followed: 33.3% of explants had GUS+ shoots in 400 mg/l cysteine and 5 mg/l PPT, 16.6% of explants had GUS+ shoots in 400 mg/l cysteine and 3.33 mg/A PPT, 8.3% of explants had GUS+ shoots in 0 mg/l cysteine and 5 mg/l PPT, 0% of explants had GUS+ shoots in 0 mg/l cysteine 3.33 mg/l PPT. Thus, adding cysteine to the co-cultivation media increases the frequency of Agrobacterium infection in the cot-node region, and results in at least a 5-fold increase in stable T-DNA transfer in newly developed shoot primordia. Other sulfhydryl-containing agents and inhibitors of the production or activity of PPO and POD also increased the frequency of transformation of soybean explants. Thus, agents of the invention reproducibly resulted in an enhanced efficiency of Agrobacterium-mediated transformation and so enhance the efficiency of producing stably transformed plants, which is particularly useful for plant tissues or cells that are difficult to transform.
Cysteine (e.g., at 400-1000 mg/l) in the solid co-cultivation medium also decreased enzymatic browning of soybean and fava bean explants. As untreated explants exhibit enzymatic browning at the wound sites on the cot-node and the cut surfaces of the hypocotyls following co-cultivation, explant wounding and infection likely activate wound and pathogen defense responses that may limit Agrobacterium-mediated T-DNA delivery to cot-node cells. The soybean cotyledon is known to be extremely responsive to pathogen attack, as exemplified by the synthesis of phytoalexins upon exposure to fungal elicitors (Boue et al., 2000). Thus, agents which inhibit the wound and pathogen defense responses on wounded and Agrobacterium-infected cot-node explants result in a reduction in enzymatic browning and tissue necrosis, increased T-DNA delivery and increased stable integration of T-DNA into the cot-node region.
In eight independent experiments, the addition of cysteine (400-1000 mg/l) resulted in: (1) an increase in the frequency of explants with at least one GUS+ focus at the cot-node from 30-100% five days post-inoculation, (2) an increase in the number of GUS+ foci per explant five days post-inoculation, (3) a 3.6-fold increase in stable T-DNA integration after 28 days, (4) a 5-fold increase in GUS+ shoot primordia after 28 days, and (5) a 2-fold increase in production of transgenic plants. Increases in T-DNA transfer also resulted from the addition of
D-cysteine, cystine, glutathione, dithiothreitol, sodium thiosulfate, and two metal chelators, bathocuproine disulfonic acid and bathophenanthroline disulfonic acid, and thus ultimately increases transgenic plant production. Preferably, the agent results in an increased stable transformation efficiency, for example, at least an increase of 0.5 to 50%, more preferably at least an increase of 2% or more, e.g., 3%, 5%, 10%, 15%, 20% or more.
Also provided is a method for transforming a plant cell, part or tissue in which the plant cell, part or tissue, e.g., apical meristem, is contacted with DNA, e.g., using a particle gene gun, and at least one agent of the invention so as to yield a transformed plant cell, part or tissue. Then a transformed plant cell, part or tissue is identified. Preferably, the addition of the agent to the plant cell, part or tissue results in an increased transformation efficiency relative to a plant cell, part or tissue which is contacted with DNA but not with the agent.
The invention also provides a method for transforming legumes. The method comprises contacting a wounded cotyledon explant from a legume seedling with an Agrobacterium containing DNA to be introduced into the explant and at least one agent of the invention so as to yield transformed explant tissue. The cotyledon is wounded in the region of the axillary bud and/or cotyledonary node. Transformed explant tissue is then identified, e.g., using a phenotypic marker present on the DNA which is introduced to the explant and/or a selectable marker such as an herbicide resistance marker. Preferably, a differentiated transformed plant is regenerated from the transformed explant tissue.
Therefore, the invention includes methods of transforming plant cells or tissues, e.g., legumes such as soybean plants, as well as regeneration of transformed tissues. Either the transformation or regeneration protocols can be used separately, but together, they provide an effective method for obtaining transgenic plants, to answer the needs of commercial farming and manufacturing. Accordingly, while both the regeneration protocol, and the transformation protocol, are described separately, it should be understood that they can, and preferably are, used in combination.
The invention also provides a transformed or transgenic plant or transformed explant prepared by the methods of the invention. For example, the invention provides transformed soybean and soybean tissue prepared from a seedling cotyledon pair containing an epicotyl, axillary buds, and hypocotyl tissue, comprising a single cotyledon containing an axillary bud and associated hypocotyl segment extending from about 0.2 to about 1.5 cm below the cotyledonary node. The associated hypocotyl segment is completely separated from its adjacent hypocotyl segment attached to the remaining cotyledon, thus separating the cotyledons. The epicotyl has been removed from the cotyledon to which it is attached, and the cotyledon is wounded in the region of the axillary bud and/or cotyledonary node. The wounded cotyledon is then contacted with Agrobacterium in the presence of an agent, e.g., cysteine, which enhances Agrobacterium infection.
Also provided is a method to identify an agent that enhances the transformation of a plant cell, plant tissue or plant part by Agrobacterium. The method comprises contacting a plant cell, plant tissue or plant part with Agrobacterium containing DNA to be introduced into the explant and at least one agent so as to yield transformed explant tissue, wherein the plant cell, plant tissue or plant part is wounded. The agent is not a phenolic, e.g., acetosyringone. Then it is determined whether Agrobacterium-mediated transformation of the plant cell, part or tissue is enhanced in the presence of the agent relative to Agrobacterium-mediated transformation of a plant cell, part or tissue which is not contacted with the agent.
Also provided is a plant medium comprising an agent of the invention. For example, the invention includes aqueous, powdered or solid media for culturing, e.g., propagating, or regenerating plant tissue, e.g., apical meristems, plant cells or a plant, which media comprises at least one of the agents of the invention. The media may be employed for propagation, e.g., micropropagation, or regeneration, of untransformed or transformed plant parts, tissue or cells, including protoplasts, e.g., from sorghum or azaleas. Preferred media are those for horticultural or floracultural purposes. In one preferred embodiment of the invention, the media is employed for propagation of tissue or cells from epiphytes, e.g., bromeliads, such as orchids. In another embodiment, the medium is one other that that employed for epiphytes. In other preferred embodiments, the medium is employed to propagate protoplasts from any plant source. Preferred agents for use in the media compositions of the invention include, but are not limited to, chelators of metals required for activity of PPO and/or POD, inhibitors of the production or activity of PPO or POD, as well as sulfhydryl-containing agents, e.g., cysteine, ascorbic acid, L-cystine, sodium thiosulfate, glutathione, or any combination thereof. Preferred media compositions of the invention are non-liquid compositions, e.g., powder or crystal formulations, comprising at least one of the agents of the invention in an preferably in an amount effective to enhance plant cell, tissue or plant survivability, decrease browning of plant cells, plant tissue or plants, inhibit the production or activity of PPO or POD in the plant cells, plant tissue or plant, or any combination thereof.